Infection of the CNS of certain individuals by HIV-1 may lead to significant cognitive and motor dysfunction, entitled HIV-1-induced encephalopathy. In addition, a number of cofactors including ethanol (Etoh) ingestion may alter penetration of HIV-1 into the CNS through the blood:brain barrier (EBB). Under-standing these cofactors involved with penetration of HIV-1 into the CNS reservoir is of critical importance now in the era of HAART. Our laboratories have recently developed an in vitro BBB system comprised totally of human CNS cell-types. We have demonstrated that various viral proteins, especially HIV-1 Vpr, has profound effects on CNS-based cells leading to apoptotic death. Utilizing complementary approaches, the effects of Etoh on HIV-1 infection of the BBB and viral penetration of the CNS will be -1 virus in toto or specific dissected . It is hypothesized that Etoh may have significant effects with the HIV HIV-1 proteins in inducing the disruption of the BBB, possibly through apoptosis, as well as increasing infection of BBB-constituting cells, including microvascular endothelial cells (MVECS) and astrocytes. In Specific Aim 1, the cellular and molecular effects of Etoh on HIV-1 infection, with and without anti-retroviral agents, of critical CNS cells in the BBB will be approached. Specific viral strains in cell-free and cell- associated (e.g, infected macrophages) forms will be utilized. In Specific Aim U, effects of Etoh with selected recombinant HIV-1 proteins on disruption of this barrier will be mechanistically analyzed. Potential disruption of the BBB by Etoh's effects with MV-1 proteins will be assessed (as for S.A.1) using complementary methods, including trans-endothelial electrical resistance (TEER) and ZOI tight junction protein alterations. Etoh's effects with select recombinant Viral proteins on apoptosis of specific BBB cells, will be analyzed via TUNEL, Annexin V assays and ion channel measurements. These effects will also be probed using mutant viruses lacking specific viral regulatory and structural genes, including Vpr, Nef, gpl2O and Tat. HIV- I vector systems to express these specific proteins intracellularly will be utilized with Etoh. Finally, alterations of specific chemokine receptors on BBB cells, especially the CNS-specific chemokine receptor APJ, will be analyzed in the setting of Etoh. Effects on chemokine receptor expression, down- regulation, and intracellular calcium fluxes after binding to relevant cognate chemokines and gpl2O will be analyzed. With these inter-related specific aims, it is proposed that a series of new technologies will be combined towards determining the precise effects of Etoh on inducing alterations in the BBB with HW-l I infection of these cells.